cAMP-gated Channels Research Articles

Photodynamic Modification of Native HCN Channels Expressed in Thalamocortical Neurons

The photodynamic process requires three elements: light, oxygen, and photosensitizer, and involves the formation of singlet oxygen, the molecular oxygen in excited electronic states. Previously, we reported that heterologously expressed hyperpolarization-activated cAMP-gated (HCN) channels in excised membrane patches are sensitive to photodynamic modification (PDM). Here we extend this study to native HCN channels expressed in thalamocortical (TC) neurons in the ventrobasal (VB) complex of the thalamus and dopaminergic neurons (DA) of the ventral tegmental area (VTA). To do this, we introduced the photosensitizer FITC-cAMP into TCs or DAs of rodent brain slices via a whole-cell patch-clamp recording pipette. After illumination with blue light pulses, we observed an increase in the voltage-insensitive, instantaneous Iinst component, accompanied by a long-lasting decrease in the hyperpolarization-dependent Ih component. Both Ih and the increased Iinst after PDM could be blocked by the HCN blockers Cs+ and ZD7288. When FITC and cAMP were dissociated and loaded into neurons as two separate chemicals, light application did not result in any long-lasting changes of the HCN currents. In contrast, light pulses applied to HCN2-/- neurons loaded with FITC-cAMP generated a much greater reduction in the Iinst component compared to that of WT neurons. Next, we investigated the impact of the long-lasting increases in Iinst after PDM on the cellular physiology of VB neurons. Consistent with an upregulation of HCN channel function, PDM elicited a depolarization of the resting membrane potential (RMP). Importantly, Trolox-C, an effective quencher for singlet oxygen, could block the PDM-dependent increase in Iinst and depolarization of the RMP. We propose that PDM of native HCN channels under physiological conditions may provide a photodynamic approach to alleviate HCN channelopathy in certain pathological conditions.

State-dependent and site-directed photodynamic transformation of HCN2 channel by singlet oxygen.

Singlet oxygen ((1)O2), which is generated through metabolic reactions and oxidizes numerous biological molecules, has been a useful tool in basic research and clinical practice. However, its role as a signaling factor, as well as a mechanistic understanding of the oxidation process, remains poorly understood. Here, we show that hyperpolarization-activated, cAMP-gated (HCN) channels--which conduct the hyperpolarization-activated current (Ih) and the voltage-insensitive instantaneous current (Iinst), and contribute to diverse physiological functions including learning and memory, cardiac pacemaking, and the sensation of pain--are subject to modification by (1)O2. To increase the site specificity of (1)O2 generation, we used fluorescein-conjugated cAMP, which specifically binds to HCN channels, or a chimeric channel in which an in-frame (1)O2 generator (SOG) protein was fused to the HCN C terminus. Millisecond laser pulses reduced Ih current amplitude, slowed channel deactivation, and enhanced Iinst current. The modification of HCN channel function is a photodynamic process that involves (1)O2, as supported by the dependence on dissolved oxygen in solutions, the inhibitory effect by a (1)O2 scavenger, and the results with the HCN2-SOG fusion protein. Intriguingly, (1)O2 modification of the HCN2 channel is state dependent: laser pulses applied to open channels mainly slow down deactivation and increase Iinst, whereas for the closed channels, (1)O2 modification mainly reduced Ih amplitude. We identified a histidine residue (H434 in S6) near the activation gate in the pore critical for (1)O2 modulation of HCN function. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iinst induced by photodynamic modification. Our study provides new insights into the instantaneous current conducted by HCN channels, showing that modifications to the region close to the intracellular gate underlie the expression of Iinst, and establishes a well-defined model for studying (1)O2 modifications at the molecular level.

State Dependent and Site Directed Photodynamic Transformation of HCN2 Channel by Singlet Oxygen

Singlet oxygen (1O2) is a little known signaling factor but a useful tool for both basic research and clinical practice. 1O2 is the central player in the Chromophore Assisted Light Inactivation (CALI) for ablating the function of protein molecules or cells and the Photodynamic therapy (PDT) for treating cancer and other diseases. However, most of the studies on 1O2 have been carried out at cell and tissue levels and yielded little information about 1O2 modification at molecular level. Here we report that the hyperpolarization-activated cAMP-gated (HCN) channels are sensitive to 1O2 modification. To increase the site-specificity of 1O2 generation, fluorescein conjugated cAMP (FITC-cAMP) that specifically binds to the HCN channels or in-frame inserted singlet-oxygen-generator (SOG) protein was used as photosensitizer. Laser pulses in milliseconds transformed the channel biophysics by slowing down the channel deactivation and increasing the voltage-insensitive component in the macroscopic current. The dependence on dissolved oxygen in the solutions, the inhibitory effect by a 1O2 scavenger and the results with HCN2-SOG fusion protein supported the involvement of 1O2. Intriguingly, 1O2 modification of HCN2 channel was state-dependent and had distinct effects on the open and closed channels. Following this insight, we located a critical Histidine residue (H434) near the activation gate in the pore. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iins by the photodynamic modification. This study provides insights into the instantaneous current conducted by HCN channel and establishes a well-defined model for studying 1O2 modification of ion channel proteins.

Local and Global Interpretations of a Disease-Causing Mutation near the Ligand Entry Path in Hyperpolarization-Activated cAMP-Gated Channel

Hyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entrypath. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket.

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin.

A(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined. HEK 293 cells stably transfected with human A(2B) adenosine receptors (HEK-A(2B) ) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye. In HEK-A(2B) cells, the selective A(2B) receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G(i) or G(o) proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK. Activation of A(2B) adenosine receptors reduced superoxide generation from mitochondrial complex I through G(i/o) , ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules

Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca2+-permeable channels and Ca2+-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca2+-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca2+-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel.

Plasma membrane Ca2+‐ATPase in the cilia of olfactory receptor neurons: possible role in Ca2+ clearance

Olfactory sensory neurons respond to odorants increasing Ca(2+) concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP-gated channels, activating Ca(2+)-dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide-gated channels for cAMP. It has been shown that a Na(+)/Ca(2+) exchanger (NCX) extrudes Ca(2+) from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca(2+)-ATPase (PMCA), and show the first evidence supporting a role in Ca(2+) removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside-out cilia membrane vesicles transported Ca(2+) in an ATP-dependent fashion. PMCA activity was potentiated by luminal Ca(2+) (K(0.5) = 670 nm) and enhanced by calmodulin (CaM; K(0.5) = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca(2+) transport, as expected for a CaM-modulated PMCA. The relaxation time constant (tau) of the Ca(2+)-dependent Cl(-) current (272 +/- 78 ms), indicative of luminal Ca(2+) decline, was increased by CE (2181 +/- 437 ms), by omitting ATP (666 +/- 49 ms) and by raising pH (725 +/- 65 ms), suggesting a role of the pump on Ca(2+) clearance. Replacement of external Na(+) by Li(+) had a similar effect (tau = 442 +/- 8 ms), confirming the NCX involvement in Ca(2+) extrusion. The evidence suggests that both Ca(2+) transporters contribute to re-establish resting Ca(2+) levels in the cilia following olfactory responses.

Activation of olfactory cyclic‐nucleotide gated channels revisited

Ion channels that are gated by cyclic nucleotides (CNG channels) have been best studied for their roles in visual and olfactory sensory transduction. Sensory stimulation of vertebrate photoreceptors by light and olfactory receptor neurones (ORNs) by odorants activates 7-transmembrane receptors, and triggers, via a G protein, changes in the intracellular concentration of second messenger cyclic nucleotides. In the case of photoreceptors, the G protein Gt stimulates phosphodiesterase leading to the hydrolysis of cGMP, which in turn promotes closure of cGMP-gated channels and hyperpolarization. In the case of ORNs, the G protein Golf stimulates adenylyl cyclase to produce cAMP, which promotes opening of cAMP-gated channels and depolarization. The ability of CNG channels to be gated by cyclic nucleotides is therefore crucial for organisms to monitor their visual and odorant environments.

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells.

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.

Getting Used to a Smell

In vertebrate olfactory neurons, odor molecules stimulate the opening of cyclic nucleotide-gated channels (CNGs). The resulting influx of Ca 2+ ions also triggers a negative-feedback mechanism in which channel activity is inhibited when bound to a Ca 2+ -calmodulin (CaM) complex. This mechanism promotes olfactory adaptation and allows animals to evaluate the odor environment continually. Two groups have determined that two of the channel's three subunits are required for odor adaptation. Munger et al. show that channels from mice lacking the CNGA4 subunit exhibited slower Ca 2+ -CaM-mediated inhibition. Bradley et al. have used a heterologous expression system to show that both the CNGA4 and CNGB1b subunits facilitate Ca 2+ -CaM binding to the open state of channel. S. D. Munger, A. P. Lane, H. Zhong, T. Leinders-Zufall, K.-W. Yau, F. Zufall, R. R. Reed, Central role of the CNGA4 channel subunit in Ca 2+ -calmodulin-dependent odor adaptation. Science 294 , 2172-2175 (2001). [Online Journal] J. Bradley, D. Reuter, S. Frings, Facilitation of calmodulin-mediated odor adaptation by cAMP-gated channel subunits. Science 294 , 2176-2178 (2001). [Online Journal]

Facilitation of calmodulin-mediated odor adaptation by cAMP-gated channel subunits.

Calcium (Ca2+) influx through Ca2+-permeable ion channels plays a pivotal role in a variety of neuronal signaling processes, and negative-feedback control of this influx by Ca2+ itself is often equally important for modulation of such signaling. Negative modulation by Ca2+ through calmodulin (CaM) on cyclic nucleotide-gated (CNG) channels underlies the adaptation of olfactory receptor neurons to odorants. We show that this feedback requires two additional subunits of the native olfactory channel, CNGA4 and CNGB1b, even though the machinery for CaM binding and modulation is present in the principal subunit CNGA2. This provides a rationale for the presence of three distinct subunits in the native olfactory channel and underscores the subtle link between the molecular make-up of an ion channel and the physiological function it subserves.

Reconstituted Ion Channels of Frog Fungiform Papilla Cell Membrane

Abstract We identified a Cl− channel, two K+ channels and a cAMP-gated channel which were isolated from bullfrog fungiform papilla cell membranes and incorporated into phospholipid bilayers using the tip-dip method. The 156 pS channels were inhibited by 100 μM 4, 4′-diisothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and displayed the reversal potential identical to the equilibrium potential of Cl−, it was identified as a Cl− channel. Two types of K+ channel had unitary conductances of 79 and 43 pS, which may correspond to those of Ca2+-activated and cAMP-blockable K+ channels observed in isolated intact frog taste cell membranes, respectively. These results suggest that the tip-dip method is useful for stable investigation of the properties of ion channels already identified in the taste cell. Furthermore, the 23 pS channels were newly found and were activated directly by internal cAMP as cyclic nucleotide-gated (CNG) nonselective cation channels established in olfactory receptor cells. Thus, our results...

Role of the cAMP Signaling Pathway in the Regulation of Gonadotropin-Releasing Hormone Secretion in GT1 Cells

Development of a line of highly differentiated neuronal cells (GT1 cells) that secrete gonadotropin-releasing hormone (GnRH) has permitted studies on the signaling mechanisms involved in regulating secretion of GnRH. This study focused on the pathways coupling GnRH secretion to elevated levels of cyclic adenosine monophosphate (cAMP) in the GT1 cell line. Two mechanisms were examined: a direct action of increased cAMP by means of cyclic nucleotide-gated (CNG) cation channels and an indirect action through activation of cAMP-dependent protein kinase A (PKA). Patch-clamp experiments were carried out on two lines of GT1 cells in both the on-cell and excised patch configurations. Treatment of perifused GT1 cells with dopamine led to a substantial rise in cAMP levels, which resembled accumulation of intracellular cAMP in GT1 cells exposed to dopamine. GT1 cells were found to express the three CNG subunits present in olfactory neurons: CN2, CNG4.3, and CNG5. The cells possessed functional cAMP-gated cation channels. Activation of PKA did not seem to be required for increased cAMP to stimulate the release of GnRH. Indeed, inhibiting PKA activity was associated with increased basal GnRH secretion. PKA inhibited adenylyl cyclase activity, probably by suppressing the cellular expression of adenylyl cyclase V. Stimulation of GnRH release by increased cAMP seems to result from neuronal depolarization triggered by increased conductance of cations by cAMP-gated cation channels. Activation of PKA may be a negative-feedback means of lowering cAMP levels. The resultant oscillations in cAMP could provide a basis for timing the pulsatile release of GnRH. These findings can be tested in vivo by using transgenic methods to examine the role of cAMP signaling in regulating pulsatile GnRH release in the rat. Proc Natl Acad Sci U S A 2000;97:1861–1866

Cyclic AMP activates Ras.

In addition to protein kinase A (PKA), cAMP regulates the activity of cAMP-gated channels and Rap1-specific guanine nucleotide exchange factors. We tested the hypothesis that the targets of cAMP might also include regulators of the Ras protooncogene. In rat thyroid cells, thyrotropin (TSH) stimulates proliferation through a cAMP-mediated pathway that requires Ras activity. Interference with Ras impairs DNA synthesis stimulated by TSH as well as cAMP elevating agents and analogs, demonstrating that the requirement for Ras lies down-stream of cAMP. Although cAMP stimulates proliferation, microinjection of the purified PKA catalytic subunit failed to do so, suggesting that factors in addition to PKA are required for cAMP-stimulated cell cycle progression. When added to thyroid cells expressing human Ha-Ras, TSH rapidly and markedly increased the proportion of GTP-bound Ras. Ras activity was increased within 1 min of TSH addition, maximal at 5-15 min, and declined to basal levels 30-60 min after hormone treatment. Cyclic AMP elevating agents elicited similar effects on Ras, indicating that TSH activates Ras through a cAMP-mediated pathway. Although cAMP-mediated, Ras activation by TSH and cAMP was independent of PKA activity. Moreover, cAMP-stimulated Ras activation was not impaired by tyrosine kinase inhibitors. These results indicate that cAMP activates targets in addition to PKA in thyroid cells, and that these targets may include regulators of Ras. The ability of cAMP elevating agents to activate Ras in addition to PKA may explain the inability of the PKA catalytic subunit to stimulate DNA synthesis in thyroid cells.

Calcium mediates the NO-induced potassium current in toad and rat olfactory receptor neurons.

Nitric oxide (NO) activates a K(+) current in dissociated amphibian olfactory receptor neurons. Using the patch-clamp technique in its whole-cell mode and stimulation with puffs of the NO-donor sodium nitroprusside, we further studied this effect and show that it was sensitive to the K(+)-channel blockers tetraethylammonium and iberiotoxin, indicating the activation of a Ca(2+)-dependent K(+) conductance. The Ca(2+)-channel blockers nifedipine and cadmium abolished the NO-induced current, and lowering external Ca(2+) reduced it significantly. Ca(2+) imaging showed a transient fluorescence increase upon stimulation with NO, and after blockade of K(+) currents, an NO-induced inward current could be measured, suggesting that the activation of the Ca(2+)-dependent K(+) conductance is mediated by Ca(2+) influx. LY83583, a blocker of the ciliary cAMP-gated channels, did not affect the current, and experiments with focal stimulation indicated that the effect is present in the soma, therefore Ca(2+) is unlikely to enter via the transduction channels. Finally, we show that NO exerts an effect with similar characteristics on olfactory receptor neurons from the rat. These data represent the first evidence that NO activates a Ca(2+)-dependent K(+) conductance by causing a Ca(2+) influx in a sensory system, and suggest that NO signaling plays a role in the physiology of vertebrate olfactory receptor neurons.

Olfactory Signal Transduction. Main Stream and Modulation.

Olfactory signal transduction starts at the ciliary membrane of the receptor cells located in the olfactory epithelium. This transduction is triggerd by the G-protein-coupled seven transmembrane receptor, which in turn activates adenylyl cyclase-cAMP second messenger system. These chemical reactions finally lead to a sequential opening of cAMP-gated channel and Ca-activated Cl channel. Olfactory sensation is known to exhibit strong adaptation to the applied smell environment. This adaptation is regulate by a Ca-feedback to the cAMP-gated channel.

Calcium signalling and regulation in olfactory neurons

The odorant-induced Ca(2+) increase inside the cilia of vertebrate olfactory sensory neurons controls both excitation and adaptation. The increase in the internal concentration of Ca(2+) in the cilia has recently been visualized directly and has been attributed to Ca(2+) entry through cAMP-gated channels. These recent results have made it possible to further characterize Ca(2+)'s activities in olfactory neurons. Ca(2+) exerts its excitatory role by directly activating Cl(-) channels. Given the unusually high concentration of ciliary Cl(-), Ca(2+)'s activation of Cl(-) channels causes an efflux of Cl(-) from the cilia, contributing high-gain and low-noise amplification to the olfactory neuron depolarization. Moreover, in combination with calmodulin, Ca(2+) mediates odorant adaptation by desensitizing cAMP-gated channels. The restoration of the Ca(2+) concentration to basal levels occurs via a Na(+)/Ca(2+) exchanger, which extrudes Ca(2+) from the olfactory cilia.

Impaired Odor Adaptation in Olfactory Receptor Neurons after Inhibition of Ca2+/Calmodulin Kinase II

Odor adaptation in vertebrate olfactory receptor neurons (ORNs) is commonly attributed to feedback modulation caused by Ca(2+) entry through the transduction channels, but it remains unclear and controversial whether this Ca(2+)-mediated adaptation resides in the cAMP-gated channel alone or whether other molecules of the transduction cascade are modulated as well. Attenuation of adenylyl cyclase activity by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has also been proposed as a mechanism for adaptation. To test this in intact ORNs, we have compared the properties of adaptation induced by a sustained (8 sec) or brief (100 msec) odor stimulus. Although adaptation induced by both types of stimuli occurs downstream from the odor receptors and is Ca(2+)-dependent, only adaptation induced by a sustained pulse involves alterations in the odor response kinetics, consistent with a reduction in the rate of adenylyl cyclase activation. By disrupting CaMKII to block adenylyl cyclase attenuation using a specific peptide inhibitor of CaMKII, autocamtide-2-related inhibitory peptide (AIP), we show that this reaction is necessary for odor adaptation in vivo. With CaMKII disrupted, adaptation induced by a sustained stimulus is significantly impaired: the onset rate of adaptation is decreased by threefold, and the recovery rate from adaptation is increased by up to sixfold. In contrast, adaptation induced by a brief odor pulse is unaffected, demonstrating that the effect of AIP must be highly specific. The results indicate that CaMKII controls the temporal response properties of ORNs during odor adaptation. We propose that CaMKII plays a prominent role in odor perception.

High-gain, low-noise amplification in olfactory transduction

It is desirable that sensory systems use high-gain, low-noise amplification to convert weak stimuli into detectable signals. Here it is shown that a pair of receptor currents underlying vertebrate olfactory transduction constitutes such a scheme. The primary receptor current is an influx of Na+ and Ca2+ through cAMP-gated channels in the olfactory cilia. External divalent cations improve the signal-to-noise properties of this current, reducing the mean current and the current variance. As Ca2+ enters the cilium, it gates Cl- channels, activating a secondary depolarizing receptor current. This current amplifies the primary current, but introduces little additional noise. The system of two currents plus divalent cations in the mucus produces a large receptor current with very low noise.

Mechanism of odorant adaptation in the olfactory receptor cell.

Adaptation to odorants begins at the level of sensory receptor cells, presumably through modulation of their transduction machinery. The olfactory signal transduction involves the activation of the adenylyl cyclase/cyclic AMP second messenger system which leads to the sequential opening of cAMP-gated channels and Ca2+-activated chloride ion channels. Several reports of results obtained from in vitro preparations describe the possible molecular mechanisms involved in odorant adaptation; namely, ordorant receptor phosphorylation, activation of phosphodiesterase, and ion channel regulation. However, it is still unknown whether these putative mechanisms work in the intact olfactory receptor cell. Here we investigate the nature of the adaptational mechanism in intact olfactory cells by using a combination of odorant stimulation and caged cAMP photolysis which produces current responses that bypass the early stages of signal transduction (involving the receptor, G protein and adenylyl cyclase). Odorant- and cAMP-induced responses showed the same adaptation in a Ca2+-dependent manner, indicating that adaptation occurs entirely downstream of the cyclase. Moreover, we show that phosphodiesterase activity remains constant during adaptation and that an affinity change of the cAMP-gated channel for ligands accounts well for our results. We conclude that the principal mechanism underlying odorant adaptation is actually a modulation of the cAMP-gated channel by Ca2+ feedback.